The trophoblast cells that form the fetal portion of the human placenta rapidly gain access to the maternal circulation by transiently exhibiting certain tumor-like properties. Thus, during early pregnancy fetal cytotrophoblast cells invade the uterus and its arterial network. This process peaks in the twelfth week of pregnancy and declines rapidly thereafter, suggesting that the invasive behavior of the cytotrophoblast cells is closely regulated. Currently, little is known about the actual mechanisms involved. Since the process of human trophoblast invasion cannot be studied in vivo, we have devised a culture system that reproduces many of these temporally regulated events. Recently, we showed that first trimester human cytotrophoblast cells, but not those isolated from second and third trimester placentas, invaded the extracellular matrices on which they were plated. During this invasive period the first trimester cells expressed a unique and complex pattern of metalloproteinases that was not expressed from the second trimester onward. The purpose of the proposed experiments is to determine whether the metalloproteinases act alone, or as part of a proteinase cascade, during trophoblast invasion. We will also isolate the relevant proteinases and determine their substrate specificities. Finally, we will investigate elements contributing to metalloproteinase regulation. We have three specific goals. First, we will study metalloproteinase function during cytotrophoblast invasion by: a) employing the zymogen, substrate gel technique to determine whether a variety of metalloproteinase inhibitors affect cytotrophoblast metalloproteinase activity; b) testing the ability of effective metalloproteinase inhibitors thus identified to perturb human cytotrophoblast invasion in vitro; and c) using inhibitors of other classes of proteinases that may participate in a proteinase cascade to perturb cytotrophoblast invasion in vitro. Second, we will isolate and characterize metalloproteinases functioning during trophoblast invasion by: a) chromatography on commercially available matrices and/or affinity matrices; b) assaying the substrate specificity of the purified metalloproteinases using extracellular matrix molecules that are most abundant in the placenta and/or the placental bed; and c) using peptide sequences of the purified proteinases for comparison with sequences of other known metalloproteinases and for synthesizing oligonucleotide probes to be used in the genetic analyses. Third, we will examine, using immunological and genetic means, the developmental regulation of cytotrophoblast metalloproteinases and proteinase inhibitors by: a) producing polyclonal and monoclonal antibodies against these antigens; b) using immunoblotting and immunolocalization to study their time synthesis within the placenta and placental bed; and c) performing mRNA analysis of human placenta to study their expression. Studying the factors that regulate trophoblast invasion will contribute substantially to our understanding of disorders that are related to defects in this process such as the abnormally deficient invasion that occurs in preeclampsia or the abnormally extensive invasion that occurs in choriocarcinoma.